CN1207131A - Recombinant fusion proteins to growth hormone and serum albumin - Google Patents
Recombinant fusion proteins to growth hormone and serum albumin Download PDFInfo
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- CN1207131A CN1207131A CN96199466A CN96199466A CN1207131A CN 1207131 A CN1207131 A CN 1207131A CN 96199466 A CN96199466 A CN 96199466A CN 96199466 A CN96199466 A CN 96199466A CN 1207131 A CN1207131 A CN 1207131A
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Abstract
Fusion proteins of albumin and growth hormone, or fusions of variants of either, are secreted well in yeast and have increased serum and storage stability.
Description
The present invention relates to the protein product in recombination fusion protein, tethelin (GH), serum albumin and the yeast.
Human serum albumin (HSA) is one to have 585 amino acid whose protein, and it is an important component part of osmotic pressure in the serum, and plays a part endogenous and carrier exogenous ligand.At present, used clinically HSA extracts from people's blood plasma.The production of reorganization HA (rHA) is open in EP 330 451 and EP 361 991 in the microbe.
Albumin is essential feature as a kind of effect and inert nature thereof of carrier molecule to the transporter that it is used as a stablizer and polypeptide.Albumin is as the component of fusion rotein, and is to stablize other proteinic application at WO 93/15199, open among WO 93/15200 and the EP 413 622.It is also open in EP 399 666 that the N-end fragment of HSA is fused to the purposes that polypeptide gets on.Being fused to that said polypeptide gets on is method by genetic manipulation, makes the DNA of coding HSA or its fragment be connected with the DNA of the said polypeptide of encoding.Then, transform or the transfection appropriate host with this fusion nucleus thuja acid, thereby make suitable plasmid expression fusion polypeptide.Nomura etc. once attempted the apo E one-tenth of expressing human in yeast saccharomyces cerevisiae and the segmental fusion rotein of HSA or HSA in nineteen ninety-five, and utilized the presequence direct secretion of HSA.Find when and the HSA total length cause excretory fusion rotein amount low (maximum production is every liter of 6.3mg) in the medium when merging, and during with 1 to 198 aminoacid sequence of HSA or 1 to 390 aminoacid sequence fusion, cause fusion rotein not to be secreted in the medium.
Human growth hormone (seeing Strobl and Thomas summary in 1994) is made up of 191 amino acid whose single peptides, and its inside forms crosslinked by two disulfide linkage.Each hGH molecule can combine with two hGH acceptor molecules, and (Cunningham etc. 1991 so that signal conducts; De Vos etc. 1992).The C-of hGH molecule is terminal with relevant in conjunction with first acceptor molecule, but the terminal bonded degree of correlation with acceptor of its N-is unknown.This hormone is by the vertical leaf glandular secretion of the precursor under the hypothalamus control, and it causes large-scale growth effect in the body.Clinically, hGH is used for the treatment of ateliosis, the Childhood chronic renal insufficiency, the fracture and the burn.The production method of the hGH that is used for the treatment of now is to extract from people's pituitary gland, perhaps recombinant expressed in the disclosed intestinal bacteria (E.Coli) in EP 127 305 (Genentech), perhaps be recombinant expressed (Zeisel etc., 1992) in the mammalian cell cultivation.
In addition, hGH has expressed (Tokunaga etc., 1995) in yeast cell, and this organism can provide a kind of flexible form of the method that discloses among the EP 60 057 (Genentech).Tsiomenko etc. have reported that homing sequence before the yeast MF α-1 secretes the effect among the hGH in yeast.The fore-end (pre-portion) of this leading sequence combined with the hGH gene cause the hGH accumulation in pericentral siphon and the vacuole outside, the expression that then causes being positioned intracellular non-glycosylated precursor that engages of the fore portion of leader sequence (pro-portion) and hGH gene.Only all combine, hGH is secreted in the substratum with the hGH gene at leader sequence two portions.Other secretion signal (presequence) is helpless to hGH equally and is secreted in the substratum, unless with the former sequence (pro sequence) of yeast self, illustrate that this former sequence is crucial to the secretion of hGH in the yeast.
In human body, hGH is secreted in the blood with the form of pulse, and its transformation period in working cycle is less than 20 minutes (Haffner etc., 1994).The removing of this hormone is mainly by the metabolism in liver and the kidney, and the removing speed among the adult is faster than children (Kearns etc., 1991).The insufficient treatment of hGH generally continues six to 24 months, carries out per week intramuscular injection therebetween three times or every day subcutaneous injection.This frequent methods of treatment is essential, because the transformation period of this molecule is short.
Poznansky etc. 1988 are by puting together into the serum albumin (HSA) in hGH and pig source or people source the transformation period that one sizable method of puting together body (about 180KD) has prolonged Porcine somatotropin.The chemical reaction that utilizes the linking agent glutaraldehyde to carry out makes average per two albumin molecules and six tethelin molecules put together.The transformation period of body in mouse of puting together of the 180KD that is produced reaches 2 to 3 hours, and the transformation period of tethelin in mouse that does not have to put together has only five minutes.Determination of activity shows: it is all active in external maintenance to put together body, even may increase activity, but non-activity in vivo.
The present invention relates to close with an albumin molecule, or its mutation or fragment and a tethelin molecule, or its mutation or fragment fusion, the tethelin that formed fusion rotein does not more merge has prolonged circulating half-life.For simplicity, only mention human albumin (HA) and human growth hormone (hGH), but other vertebrate albumin and tethelin include interior.Preferably, contain HA, or its mutation or segmental N-terminal portions and hGH, or its mutation, or the fusion rotein of segmental C-terminal portions can make all possible negative effect of receptors bind minimize.Perhaps, contain HA, or an one mutation, or its segmental C-terminal portions and hGH, or an one mutation, or the effect of the same tool signal conduction of the fusion rotein of its segmental N-terminal portions.Usually, this polypeptide only has the zone in a HA source and the zone in a GH source.
In addition, the fusion rotein among the present invention merges between part at two and also has a joint peptide so that bigger physical isolation (physical separation) is arranged between fusion rotein two components, thereby make the hGH part most possibly with the hGH receptors bind.The joint peptide is made up of amino acid makes that it is snappiness or inflexible.
Joint sequence can be by a kind of proteolytic enzyme or chemical process cutting, to produce the relevant part of tethelin.Preferably, this proteolytic enzyme can be by the natural generation of host, as: Saccharomyces cerevisiae Proteinase K ex2 or suitable proteolytic enzyme.After this, the present invention also further provides polynucleotide by expressing polypeptide in the code book invention in an appropriate host with preparation tethelin, or its mutation, or its segmental method and the cutting joint that can cut are to produce GH-type mixture and obtain the method for GH-type mixture with purer form from host's substratum.
Find: the stability of the polypeptide among the present invention in solution is apparently higher than hGH.The latter stores rapid inactivation after month under 4 ℃ in solution.Present commercially available hGH is a lyophilisate.
Suitably, fusion polypeptide is by yeast, microorganism, and as bacterium, human cell line or yeast produce with the form secretion of recombinant molecule.Preferably, this polypeptide can be secreted from the host.Find: the sequence of the hGH that will encode be fused to 5 of coding HA sequence '-end or 3 ' end, yeast all can be secreted this fusion rotein, and need not the former sequence (pro sequence) that a primary yeast is originated.This discovery is surprising, because other workers find that it is essential that the former sequence in yeast source is effectively secreted hGH to yeast.As: nineteen nineties and 1991 such as Hiramitsu find that respectively the N-end parts of preceding former homing sequence (pre-pro leader) the Central Plains sequence (pro sequence) of Mucor pusillus (Mucor pusillus) bacterial strain rennin is important.Other author with MF α-1 signal secretion hGH the time, always comprises the former sequence of MF α-1.This former sequence is considered to help as a kind of intramolecular molecular chaperones the folding process of hGH.The present invention shows that the fragment of HA or HA can exercise similar function.
Thereby, a specific embodiment among the present invention comprises: the DNA construct of direct secretion coded signal sequence effectively in the yeast, especially yeast source signal sequence (signal sequence that homology is particularly arranged with yeast host), with the fusion molecule of a first aspect of the present invention, wherein there is not the former sequence in yeast source to exist between signal and sophisticated polypeptide.
The signal yeast of Saccharomyces cerevisiae (S.cerevisiae) saccharase comes preferred embodiment of source signal.
Do not consider that Poznansky etc. utilized Chemical Crosslinking Methods to connect two the sort of bodies of puting together of preparation respectively in 1988.
Albumin or hGH can be respectively the variants of normal HSA/rHA (hereinafter referred to as " HA ") or hGH.In these " variants ", be included in the insertion of conservative region or non-conservative region, disappearance and replacement, but these variations can not obviously change (oncotic) of albuminous one or more swelling useful part combination and nonimmunogenic feature, or in hGH then be its non-immunogenic and can in conjunction with and activate the ability of hGH acceptor.Especially, the polymorphism variant of natural generation in human albumin and the human albumin fragment is included, as: those disclosed fragment in EP 322 094 (be called HA (1-n), wherein n refers to 369 to 419).Albumin or tethelin can come from any vertebrates, particularly any Mammals, as: people, ox, sheep, pig, chicken (hen), or salmon.Albumin in the fusion rotein can come from different animals respectively with the GH part.
" conservative property surrogate " is with referring to such as glycine (Gly), L-Ala (Ala); Xie Ansuan (Val), Isoleucine (Ile), leucine (Leu); Aspartic acid (Asp), L-glutamic acid (Glu); N (Asn), glutaminase (Gln); Serine (Ser), Threonine (Thr); Methionin (Lys), arginine (Arg); And phenylalanine (Phe), the replacement in these groups of tyrosine (Tyr).As long as think in this area that it is usual allowing to insert and delete, the normal HA of these variants and same length or hGH have at least 75% sequence homology (tool 80% at least preferably, 90%, 95%, or 99% homology), and than the normal HA or the hGH of other length have more homology.Put it briefly, a HA variant is 100 amino acid lengths of tool at least, preferably have 150 amino acid lengths at least.This HA variant comprises at least or is made up of one of HA complete structural domain, as structural domain 1 (1-194), and 2 (195-387), 3 (388-585), 1+2 (1-387), 2+3 (195-585), or 1+3 (1-194 ,+388-585).Each structural domain itself is made up of two homology subdomains, these subdomains are called 1-105,120-194,195-291,316-387,388-491, and 512-585, comprise Methionin (Lys) 106 to L-glutamic acid (Glu) 199, the amino-acid residue of L-glutamic acid (Glu) 292 to Xie Ansuan (Val) 315 and L-glutamic acid (Glu) 492 to L-Ala (Ala) 511 districts have flexible between-the subdomain joint area.Preferably, the HA of fusion rotein part comprises the subdomain of a HA at least, or structural domain, or their conservative property is modified part.If this fusion rotein is based on subdomain, the part or all of joint that adjoins is preferably used for connecting the hGH moiety.The variant of hGH should have the GH activity, generally have ten amino acid when some authors only find four residues of tool (although same tool activity) at least, preferably have 20 amino acid at least, preferably have 50 at least, 100,150,180, or 191 amino acid longs, and preferably keep the halfcystine (Cys) of two inner disulfide linkage.
The molecular weight of fusion molecule generally is lower than 100KD among the present invention, as is lower than 90KD or 70KD.Thereby they than the 180KD of Poznansky etc. to put together body (literary composition sees before) much smaller, and be non-activity in latter's body.Under the normal circumstances, the molecular weight of this fusion rotein has 20KD at least, has 30KD or 50KD usually at least.The molecular weight size of most of fusion roteins is between 60 to 90KD.
Second major portion of the present invention provides transformed yeast to express the fusion rotein among the present invention.
Except that transformed host cell self, the present invention also considers to carry out the cultivation of these cells in a nutrient media, preferably mono-clonal (clone's homogeneous) cultivation, or a cultivation that comes from the mono-clonal culture.In case particularly polypeptide secretion has this polypeptide and these cells simultaneously in the substratum, perhaps by filtration or centrifugation method these cells are removed.
Known have many expression systems, comprise bacterium (as: intestinal bacteria (E.Coli), and subtilis (B.Subtitis)), yeast (as: Saccharomyces cerevisiae (S.cerevisiae), newborn Crewe Vickers yeast (K.lactis) and Pichia pastoris (Pichia pastoris)), filamentous fungus (as: Eurotium (Aspergillus)), vegetable cell, zooblast and insect cell.
Desirable proteins produces by convenient mode, as producing from the encoding sequence of free plasmid from being inserted into host chromosome or.
The encoding sequence that transforms desirable proteins available usual method in the yeast, as: electroporation.Method with the electroporation transformed yeast is open by Becker and Guarente, (" Enzymology method (MethodsEnzymol) " nineteen ninety, the 182nd page of the 194th phase).
The success cell transformed promptly contains the cell of DNA construct among the present invention, can be identified by the technology that people know.As, the introducing of expression construct will be grown and be produced required polypeptide in the cell.These cells are through collecting and cracking, available Southern was published in " molecular biology magazine (J.Mol.Biol) " the 98th phase method of the 503rd page in 1975, or whether Berent etc. was published in existence that " biotechnology (Biotech) " third phase method of the 208th page checks this DNA among its DNA in 1985.Perhaps, the albumen available antibodies in the supernatant detects.
Useful yeast plasmid carrier comprises pRS 403-406 and pRS 413-416, and these carriers generally can obtain from Stratagene Cloning Systerns, (Stratagene Cloning systerns, LaJolla, CA92037, USA).Plasmid pRS 403, pRS 404, and pRS 405 and pRS 406 are yeast integrative plasmid (YIp), and are combined with yeast selected marker HIS3, TRP1, LEU2 and URA3.Plasmid pRS 413-416 is yeast centromeric plasmid (YCp).
Made up and variously DNA has been operably connected to the method that carrier gets on by complementary sticky end.As, add that on dna fragmentation complementary with the aggressiveness sequence, goes thereby be inserted in the carrier DNA.Yet carrier and dna fragmentation link to form recombinant DNA molecules by form hydrogen bond between the complementary homopolymeric tail.
Synthetic linker with one or more restriction sites provides links the another kind of method that carrier gets on dna fragmentation.Dna fragmentation by the restriction enzyme enzyme liberating is handled with phage T4 archaeal dna polymerase or intestinal bacteria (E.Coli) dna polymerase i, these enzymes with their 3 '-5 ' exonuclease activity remove 3 '-protruding terminus of strand, mend with its polymerase activity then flat 3 '-recessed end.
Thereby these active associatings are used the DNA sections that has produced the flat end of tool.Then the excessive many linkers of this flat terminal sections and mole number can catalysis put down enzyme (as phage T4 dna ligase) that terminal dna molecular is connected in the presence of incubation.Therefore, reaction product is the terminal DNA sections that carries with the aggressiveness joint sequence.Then, this DNA sections with suitable Restriction Enzyme cutting, and is linked with enzyme and cut and produce on the expression vector with the compatible end of these DNA sections.
Synthetic linker commercialization with various restriction enzyme enzyme recognition sites can be by comprising that (CN USA) obtains in interior various approach for International Biotechnologies Inc, New Haven in international biotech company.
If need, as, the variant of preparation HA, a Perfected process modifying DNA of the present invention can be published in polymerase chain reaction (PCR) method of " science (Science) " the 239th phase 487-491 page or leaf with Saiki etc. in 1988.In this method, treat that the DNA of enzymatic amplification has added two special Oligonucleolide primers respectively in its both sides, and primer itself can mix among the DNA of this amplification.Said special primer can restricted property restriction endonuclease recognition site, and the known method in available this area is cloned into it in expression vector and is gone.
The host yeast demonstration genus that can be used for expressed fusion protein used among the present invention is: pichia belongs to (Pichia (Hansenula)), yeast saccharomyces cerevisiae belongs to (Saccharomyces), kluyveromyces spp (Kluyverornyces), Candida (Candida), torulopsis (Torulopsis), spore torulopsis (Torulaspora) is arranged, Schizosaccharomyces (Schizosaccharomyces), Citeromycesbaodingensis belongs to (Citeromyces), (Pachysolen), Debaryomyces (Debaromyces), the strange yeast belong of plum (Metschunikowia), red teliosporeae (Rhodosporidium), colourless basidiospore belongs to (Leucosporidium), Portugal's shape arthroderma (Botryoascus), and lock is thrown yeast belong (Sporidiobolus), Endomycopsis (Endomycopsis), and similar genus.Be selected from preferred the genus: yeast saccharomyces cerevisiae belongs to (Saccharomyces), Schizosaccharomyces (Schizosaccharomyces), and kluyveromyces spp (Kluyveromyces), pichia belongs to (Pichia) and spore torulopsis (Torulaspora) is arranged.Wherein yeast saccharomyces cerevisiae belongs in (Saccharomyces spp) genus has yeast saccharomyces cerevisiae (S.cerevisiae), S.italius and S.rouxill..Kluyveromyces spp (Kluyveromyces spp.) has in belonging to: Kluyveromyces fragilis (K.fragilis), newborn kluyveromyces (K.lactis) and kluyveromyces marxianus (K.marxianus).Suitable spore torulopsis (Torulaspora) kind that has has the Shuo Shi of Dell that spore torula (T.delbrueckii) is arranged.Pichia belongs to P.angusta (being multiform Hansenula anomala (H.polymorpha) in the past), unusual pichia (P.anomala) (being unusual Hansenula anomala (H.anomala) in the past) and Pichia pastoris (P.pastoris) in (Hansenula) (Pichia (Hansenula) spp.) genus.
The method of transformed saccharomyces cerevisiae has briefly been introduced among EP 258 067 and the WO 90/01063 at EP 251 744, and all these all incorporate the present invention into the reference form.
Suitable promotor comprises the gene with PGK1 in the yeast saccharomyces cerevisiae, GAL1 or GAL10 gene, CYC1, PH05, TRP1, ADH1, the ADH2 gene, glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase), hexokinase (hexokinase), pyruvic carboxylase (Pyruvate decarboxylase), phosphofructokinase (phosphofructokinase), triose-phosphate isomerase (triose phosphate isomerase), phosphoglucose isomerase (phosphoglucoseisomerase), glucokinase (glucokinase), α-pairing factor pheromone (α-mating factorpheromone), the relevant promotor of gene of a-pairing factor pheromone (a-mating factor pheromone), the PRB1 promotor, the GUT2 promotor, the GPD1 promotor, hybrid promoter (comprise 5 '-control region part and 5 of other promotor '-control region part heterozygosis, or with the promotor of UAS (as: promotor among the EP-A-258 067) heterozygosis).
The promotor of using in schizosaccharomyces pombe (Schizosaccharomyces pombe) of regulating and control easily is that VitB1 in the nmt gene is checked the promotor (being addressed in nineteen ninety " genetics Genetics " the 124th phase 807-816 page or leaf by Hoffman and Winstion) that type promotor (this promotor is addressed in nineteen ninety " journal of biological chemistry J.Biol.chem. " the 265th phase 10857-10864 by Maundrell) and glucose check type fbp1 gene.
Transform Pichia with the method for expression alien gene as Cregg etc. 1993, and various phillips patents are (as US 4 857 467, incorporate the present invention into the reference form) the middle description, Pichia expresses test kit commercialization, can be from Invitrogen BV, Leek, Netherlands and InvitrogenCorp., San Diego, California obtains.Suitable promotor comprises AOX1 and AOX2.
Gleeson etc. were published in " microbial genetics magazine " in 1986 and (J.Gen.Microbiol) comprise the information of Hansenula carrier and conversion in the method for the 132nd phase 3459-3465 page or leaf, and suitable promotor is MOX1 and FMD1; And EP 361991, Fleer etc. (1991) and some other come from Phone-Poulenc Rorer report and then tell people how expression foreign protein in Crewe Vickers yeast belong (Kluyveromyces spp.), and wherein suitable promotor is PGK1.
Transcription termination signal be preferably 3 of an eukaryotic gene '-the lateral order sequence, comprise suitable transcription termination signal and polyadenylic acid signal in this sequence.Suitable 3 '-the lateral order sequence is for as those natural genes that is connected on the used expression control sequenc, promptly is equivalent to promotor.Perhaps, when preferably using yeast saccharomyces cerevisiae ADH1 gene termination signal, they can be different.
Required fusion rotein can be with the initial expression of a secretor type leader sequence, and this leader sequence can be any at the effective leader sequence of selected yeast.Useful leader sequence comprises pairing factor-alpha polypeptide (MF α-1) and the heterozygosis homing sequence that comes among the EP-A-387 319 in the yeast saccharomyces cerevisiae.These homing sequences (or signal) are discharged in sophisticated albumin and are promptly cut by yeast before going in the surrounding medium.And this leader sequence comprises: disclosed yeast saccharomyces cerevisiae saccharase (SUC2) among the JP 62-096086 (mandate is 91/036516); Acid phosphatase (PH05); MF α-1, the presequence of beta-glucanase (BGL2) and lethal toxin; The glucoamylase II of S.diastaticus; The o-tilactase (MEL1) of S.carlsbergensis; Lethal toxin among the K.lactis; And the glucoamylase of candidiasis (Candida).
Fusion rotein among the present invention or its prescription can comprise non-method administration through gi tract (as subcutaneous or muscle) injection or venoclysis by any traditional method.Treatment is included in and uses single dose or compound dosage in for some time.
Fusion rotein among the present invention can use separately, preferably then uses for the form that becomes pharmaceutical preparation with one or more acceptable carriers.This carrier must be compatible with fusion rotein and can not say it is " acceptable " to receptor autophosphorylation on the deleterious meaning.Typical carrier is water or salt solution, and they need be aseptic, and do not have pyrogeneous substance.This preparation needs right and wrong immunogenic; The vaccine type preparation that comprises adjuvant is not considered.
Said preparation can dosage unit convenient form exist, and can prepare by the known method of any field of pharmacology.These methods comprise and join fusion rotein and the carrier with one or more auxiliary compositions to one step.Put it briefly, with activeconstituents and liquid phase carrier or segmentation solid phase carrier or its both closely evenly in conjunction with the preparation said preparation, then, if necessary, with product shaping.
Be suitable for the non-preparation that uses through the gi tract approach and comprise aqueous or water-free aseptic parenteral solution, this liquid contains antioxidant, damping fluid, and fungistat and solute make the perviousness of medicine equate with perviousness in the acceptor blood to be administered; Aqueous and water-free sterile suspensions (comprising suspension agent and thickening material).Said preparation can be present in the container of dosage unit or multiple doses, in the ampoule and tubule as sealing, stores under freeze-drying (lyophilization) condition, promptly adds aseptic liquid carrier before the use, as water for injection.Provisional injection liquid and suspension can prepare by sterile powder.
Preferred dosage unit preparation is those daily dosage or unit that include activeconstituents, daily sub-doses or its suitable aliquot.
Fusion rotein among the present invention is used for the treatment of any situation that shows growth hormone deficiency, as isolated growth hormone deficiency disease (isolated growth hormone deficiency), full pituitary function weakens (Panhypopituitarism), (following cranial irradiation) after the skull internal radiation (as, in treatment leukemia or cerebral tumor), Turner syndrome (Turner ' s-syndrome), Tang Shi innate stupid disease (Down ' s Syndrome), uterine growth obstacle (intrauterine growth retardation), spontaneous growth defect (idiopathic growth deficiency), chronic renal failure (chronic renal faiure), fetal rickets (achondroplasia), female infertile (female infertility) and various katabolism disorder.They also can be used for stimulating growth and (or) increase the proportion of the lean meat of domestic animal (as: ox, sheep, goat and pig).
This fusion rotein can be with insulin-like growth factor I (IGF-1) administration.
Using dosage can calculate with respect to tiring of hGH by tiring of fusion rotein, considers the transformation period that fusion rotein prolongs with respect to natural hGH this moment.The typical amounts of tethelin is that per week 0.3 is to 30.0IU/kg, as 0.9 to 12.0IU/kg/ week, if 1 year three or seven subpackage dosage are used or is more.In the fusion rotein that merges by total length HA and total length GH, mean according to the suitable dosage of unit and to use the greater weight preparation, but the frequency of administered formulation can reduce, as a week twice, once a week or still less.
The preferred embodiment of the invention will be by embodiment and is described with reference to the accompanying drawings.Wherein:
Fig. 1 shows the human growth hormone cDNA sequence of the hGH of encoding mature;
Fig. 2 shows the restriction map of pHGH1;
Fig. 3 shows the restriction map of pBST (+) and the dna sequence dna of polylinker;
Fig. 4 shows the structure of pHGH12;
Fig. 5 shows the structure of pHGH16;
Fig. 6 shows the proteic zone of HSAcDNA sequence, particularly encoding mature;
Fig. 7 shows the structure of pHGH14;
Fig. 8 shows the structure of pHGH38;
Fig. 9 shows the structure of pHGH31;
Figure 10 shows the structure (embodiment 7) of pHGH58 or pHGH59;
Figure 11 is the constructing plan of the fusion rotein of a tool transcribed spacer;
Figure 12 pharmacokinetic study result is to show mouse mainline
125The rHA-hGH of I mark compares to the clearance rate of hGH.Shown in the data of two mouse of data in being every group, comprise total radioactivity and can pass through the sedimentary radioactivity of TCA, promptly its relevant with albumen rather than with dissociate
125I is relevant.About six minutes of the removing transformation period of the hGH that is calculated, the removing transformation period of rHA-hGH fusion rotein then reaches 60 minutes, sees embodiment 3.-hGH (sum) ■-hGH (TCA precipitates number) -rHA-hGH (sum) ▲-rHA-hGH (TCA precipitates number).
The recombinant DNA process of all standards is seen narration in 1989 such as Sambrook, will indicate during different situations.The dna sequence dna of coding HSA comes from EP 201 239 disclosed cDNA.The clone of embodiment 1:hGH cDNA.
The cDNA of hGH by the method for pcr amplification from human pituitary cDNA library, obtain (catalog number (Cat.No.) is HL1097V, (and Clontech Laboratories, Inc).Two Oligonucleolide primers that is suitable for hGH cDNA amplification: HGH1 and HGH2 are synthetic with the applying biological 380B of system (Applied Biosystems380B) oligonucleotide synthesizer.
HGH1：5′-CCCAAGAATTCCCTTATCCAGGC-3′
HGH2：5′-GGGAAGCTTAGAAGCCACAGGATCCCTCCACAG-3′
HGH1 and HGH2 and hGH cDNA sequence corresponding section have the difference (Fig. 1, Martial etc., 1979) of two and three Nucleotide respectively, thereby, behind the pcr amplification, introduce an EcoRI site at 5 of cDNA '-end, 3 '-BamHI site of end introducing.In addition, there is a Hind III site among the HGH2 in hGH sequence downstream and then.
Carrying out pcr amplification with Perkin-ELmer-Cetus Thermal Cycler 9600 amplification instrument and Perkin-Elmer cetus PCR test kit is to carry out with the single stranded DNA template of the bacteriophage particles (phage particles) that is located away from the cDNA library, the template source is as follows: add 10 μ l phage splitting damping fluids (the TE damping fluid that contains 280 μ g/ml. Proteinase Ks) with cracking 10 μ l bacteriophage particles, 55 ℃ of incubations are after 15 minutes, 85 ℃ 15 minutes.After cultivating 1 minute on ice, 14, centrifugal 3 minutes of 000rpm is with precipitation phage fragment.The PCR reaction mixture contains 6 these dna profilings of μ l, the concentration of every kind of primer of 0.1 μ M and the every kind of deoxyribonucleotide that is 200 μ M.Carry out 30 PCR circulation altogether, 94 ℃ of sex change 30 seconds, 65 ℃ of annealing 30 seconds, 72 ℃ were extended 30 seconds, whenever through a circulation extension time were increased by 1 second.The single product that shows an expection size (589 bases) by the gel electrophoresis analysis reactant.
Pcr amplification product carries out purifying with Wizard PCR Preps dna purification system (Promego Corp), cuts with EcoRI and Hind III enzyme then.Further, they are cloned on PUC 19 (GIBCOBRL) plasmid of a usefulness EcoRI and Hind III enzymolysis, form pHGH1 (Fig. 2) by behind the gel electrophoresis purifying EcoRI-HindIII endonuclease bamhi.The dna sequencing result in EcoRI-Hind III district shows that this PCR product is (Martial etc., 1979) identical with the hGH sequence on sequence, just its 5 '-end and 3 '-hold to have added an EcoRI site and BamHI site respectively.The expression of embodiment 2:hGH cDNA
Phagemid pBluescribe (+) polylinker sequence (Stratagene) is substituted and forms pBST (+) (Fig. 3) by the oligonucleotide joint that the oligonucleotide annealing of inserting by two long 75-mer forms between EcoRI and HindIII site.Contain a unique N otI site (complete sequence in polylinker district is seen Fig. 3) in the new polylinker.
Contain the PRB1 promotor, the DNA of coding HSA/MF α-1 heterozygosis leader sequence, the NotI HSA expression cassette of the DNA of coding HSA and the pAYE 309 (EP 431 880) of ADH1 terminator is transformed into pBST (+) and goes up to form pHA1 (Fig. 4).Cut with Hind III enzyme the encoding sequence of HSA is removed from this plasmid, reconnect again to form pHA2 (Fig. 4).
The clone of the hGH cDNA that addresses among the embodiment 1 provides the hGH coding region of preceding 8 base pairs (bp) that lack former hGH sequence and ripe hGH sequence.Be to make up the expression plasmid of secretion hGH in the yeast, 5 of clone's hGH sequence '-the following Yeast promoter that adds of end, preceding 8 base pairs of signal peptide and this hGH sequence:
With the fragment of the Hind III-SfaNI of pHA1 by two synthetic oligonucleotide HGH3 and HGH4 be added to the EcoR1-Hind III of pHBH1 segmental 5 '-end:
HGH3：5′-GATAAGATTCCCAAC-3′
HGH4：5′-AATTGTTGGGAATCTTT-3′
The pHA2 that the Hind III fragment cloning that so forms is cut to Hind III enzyme goes up formation pHGH2 (Fig. 4), thereby hGH cDNA is placed the catchment of PRB1 promotor and HSA/MF α-1 fusion leader sequence (WO 90/01063).The NotI expression cassette that is included among the pHGH2 is cloned into the middle pHGH12 (Fig. 4) of formation of pSAC35 (Sleep etc., 1990) that the NotI enzyme is cut, and wherein the NotI expression cassette comprises the ADH1 terminator in hGH cDNA downstream.This plasmid contains whole 2 μ m plasmids, thereby has copy function and can be used for screening the LEU2 gene of transformant.
PHGH12 is incorporated among the yeast saccharomyces cerevisiae DH1 (Sleep etc., 1990) by conversion, and one transformant was grown three days for 30 ℃ in 10ml YEPD (1%w/v yeast extract, 2%w/v peptone, 2%w/v dextran).Behind centrifugal this cell, supernatant detects by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), finds to contain the protein of expectation size, and western blotting (Westernblots) shows that this albumen can be by anti-hGH antiserum(antisera) (Sigma, Poole, UK) identification.The clone and the expression of embodiment 3:HSA-hGH fusion rotein
For HSAcDNA being fused to 5 of hGH cDNA '-end, with HGH8 the EcoR1-Hind III fragment of pHA1 Hind III-Bsu 36I fragment (major part that contains HSA cDNA) and pHGH1 is connected by two oligonucleotide HGH7:
HGH7：5′-TTAGGCTTTTCCCAAC-3′
HGH8：5′-AATTGTTGGGAATAAGCC-3′
The Hind III fragment cloning that so forms is formed pHGH10 (Fig. 5) in the pHA2 that cuts with Hind III enzyme, the expression cassette of this plasmid NotI is cloned among the pSAC35 that the Not1 enzyme cuts and is formed pHGH16 (Fig. 5).
PHGH 16 is used for being transformed into yeast saccharomyces cerevisiae DB1, the methods analyst of the supernatant of cultivation among the embodiment 2.Observe the master tape of the about 88KD of a part amount size, be equivalent to the size after HA and the hGH combination.The Western trace that carries out with anti-HSA and anti-hGH antiserum(antisera) (Sigma) has confirmed the existence of two integral parts in the fusion rotein.
From culture supernatant, successively use cation-exchange chromatography, anion-exchange chromatography and this fusion rotein of gel permeation chromatography purifying.Proved the existence of the albumin sequence of expection by the amino acid sequence analysis of its N-end.
The growth in vitro hormonal activity is measured (Ealey etc., 1995) and is shown that this fusion rotein has whole hGH activity, tires and has reduced but compare it with the hGH standard.At the mouse weight increase model of a hypophysectomy (substantially according to European Pharmacopoeia (1987, monograph 556) method described) in, when using identical activity unit quantity (based on above-mentioned external test) every day, fusion protein molecule is than higher the tiring of hGH tool.Further experiment was administered once with fusion rotein in promptly per four days, showed all to use it and every day the hGH whole growth responses of tool similarly.Pharmacokinetic experiment is (promptly to the mouse administration
125The albumen of I mark) circulating half-life that shows fusion rotein has increased by ten times (Figure 12) approximately than hGH.
Substitute the heterozygosis leader sequence making up a similar plasmid with the DNA of coding yeast saccharomyces cerevisiae saccharase (SUC2) leader sequence, thus the homing sequence of coding and as follows with HSA sequence joint:
MLLQAFLFLLAGFAAKISA?↓?DAHKS......
Saccharase leader sequence HSA in being transformed into yeast saccharomyces cerevisiae DB1 after, the control of this plasmid is expressed and the degree of secretion fusion rotein and usefulness pHGH16 similar.The N-end of fusion rotein is analyzed and has been shown leader sequence accurate and effectively cutting from maturation protein.The clone and the expression of embodiment 4 hGH-HSA fusion roteins
For hGH cDNA being fused to 5 of HSAcDNA '-end (Fig. 6), at first by rite-directed mutagenesis change HSA cDNA with 5 of coding region '-end near EcoNI site of introducing.By (1987) methods such as a Kunkel single stranded DNA template and synthetic oligonucleotide LEU4 who prepares by pHA1:
LEU4:5 '-GAGATGCACACCTGAGTGAGG-3 ' finishes.Rite-directed mutagenesis with this oligonucleotide is meant that the coding region that changes HSA cDNA is so that Lys4 becomes Leu 4 (K4L).Yet this change has promptly been repaired after 5 of hGH cDNA '-end is linked sudden change pHA1 subsequently.This connection is by two oligonucleotide HGH5 and HGH6 the NotI-BamHI fragment of pHGH2 to be connected on the EcoNI-NotI fragment of sudden change pHA1.
HGH5：5′-GATCCTGTGGCTTCGATGCACACAAGA-3′
On the pSAC 35 that the NotI fragment cloning of HGH6:5 '-CTCTTGTGTGCATCGAAGCCACAG-3 ' formation is cut to the NotI enzyme to constitute pHGH14 (Fig. 7).With the method among the embodiment 2 pHGH14 is transformed among the yeast saccharomyces cerevisiae DB1, and analyzes culture supernatant.Observe the master tape that a part amount size is about 88KD, itself and sizableness after hGH and HA combine.Utilize anti--HSA and anti--Western Blot experiment that the hGH antiserum(antisera) carries out to confirm the existence of two integral parts of this fusion rotein.
Fusion rotein in the culture supernatant is successively used cation-exchange chromatography, anion-exchange chromatography and gel permeation chromatography purifying.Proved the existence of the hGH sequence of expection by the amino acid sequence analysis of its N-end.
In vitro study shows that this fusion rotein remains with the activity of hGH, but it is tired and is starkly lower than the fusion rotein that contains total length HA (1-585) N-end parts and hGH C-end parts among the embodiment 3.Embodiment 5: the structure of the expression plasmid of the hGH that merges with the HSA structural domain.
Preceding two structural domains of hGH molecule and HA (1 to 387 amino-acid residue) merge and form fusion polypeptide.By oligonucleotide HGH11 and HGH12 with the Hind III-SapI fragment of pHA1 (this fragment have HA structural domain 1 and 2 most of encoding sequence) link the N-end that forms hGH on the EcoRI-HindIII fragment of pHGH1 and merge.
HGH11：5′-TGTGGAAGAGCCTCAGAATTTATTCCCAAC-3′
HGH12：5′-AATTGTTGGGAATAAATTCTGAGGCTCTTCC-3′
So the pHA2 that cuts to Hind III enzyme of the Hind III fragment cloning that forms goes up and forms pHGH37 (Fig. 8), and the NotI expression cassette of this plasmid is exempted among the grand pSAC 35 that cuts to the NotI-enzyme.The plasmid pHGH38 (Fig. 8) that forms has an expression cassette, can be with the fusion polypeptide direct secretion in supernatant liquor after in being transformed into yeast saccharomyces cerevisiae DB1.The Western trace that utilizes anti--HSA and anti-hGH antiserum(antisera) to carry out confirms the existence of two component parts in the fusion rotein.
Priority is by cation-exchange chromatography and this fusion rotein of hydrogel permeation layer analysis method purifying from culture supernatant.The purifying protein in vitro study shows that its circulating half-life is longer than hGH, and total length HA (1-585) N-end parts is similar with the fusion rotein circulating half-life of hGH C-end parts to having among the embodiment 3.In vitro study shows that this fusion rotein remains with the activity of hGH.
Utilization will have the N-end parts of first structural domain of HA (the 1st to 194 amino-acid residue) and the fusion rotein of hGH C-end parts and be cloned among the yeast saccharomyces cerevisiae DB1 and expression therein similar in appearance to the method that describes in detail above.With anti-HSA and anti-hGH antiserum(antisera) supernatant liquor is carried out the Western engram analysis, proved the existence of two component parts in the fusion rotein.Embodiment 6: express hGH by add cleavage site between HSA and hGH.
Between the HA-hGH fusion rotein, introduce one and can be secreted hGH by the peptide sequence that Kex2 proteolytic enzyme is discerned.Utilize two oligonucleotide HGH14 and HGH15 that the sequence of one encoding serine (Ser)-leucine (Leu)-aspartic acid (Asp)-Methionin (Lys)-arginine (Arg) is introduced.
HGH14：5′-TTAGGCTTAAGCTTGGATAAAAGATTCCCAAC-3′
These are used for the Hind III-Bsu 36I fragment of pHA1 is received the EcoRI-Hind III fragment of pHGH1 HGH15:5 '-AATTGTTGGGAATCTTTTATCCAAGCTTAAGCC-3 ', are cloned into then among the pHA2 that Hind III enzyme cuts and form pHGH25 (Fig. 9).The NotI expression cassette of this plasmid is cloned among the pSAC35 that the NotI enzyme cuts forms pHGH31 (Fig. 9).PHGH31 is transformed among the yeast saccharomyces cerevisiae DB1, and SDS-PAGE analyzes culture supernatant, finds two kinds of main components of its secretion.The molecular weight of these two kinds of compositions is about respectively and the suitable 66KD of (total length) HA, is equivalent to the 22KD of (total length) hGH, and an outer available Kex2 proteolytic enzyme of display body or a similar active protease cut this fusion rotein.The Western trace that carries out with anti-HSA and anti-hGH antiserum(antisera) has proved the existence of these two kinds of separated components.To hGH N-terminal sequence analytical proof partly accurately and effectively cutting from the HA part.
Culture supernatant successively by anion-exchange chromatography and gel permeation chromatography with purifying hGH part.The in vitro study of the hGH of this purifying shows that this albumen has active and whole the tiring of tool.
Utilize similar strategy, to contain first structural domain of HA (the 1st to 194 amino-acid residue), perhaps contain preceding two structural domains of HA (the 1st to 387 amino-acid residue), and the fusion rotein that is connected to the sequence that can be discerned by Kex2P proteolytic enzyme and hGH cDNA is cloned among the yeast saccharomyces cerevisiae DB1 and is expressed.The Western trace that culture supernatant is carried out with anti-HSA and anti-hGH antiserum(antisera) has proved the existence of these two kinds of independent components.Embodiment 7:HSA and hGH are by the fusion of a flexible joint sequence
By oligonucleotide HGH 16, HGH 17, HGH 18, the clone of HGH 19, introduce flexible joint between the fusion rotein of HSA and hGH, this joint contains [glycine (Gly)-glycine (Gly)-glycine (Gly)-glycine (Gly)-Serine (Ser)] n repeating unit (wherein n can be 2 or 3).
HGH16：5′-TTAGGCTTAGGTGGCGGTGGATCCGGCGGTGGTGGAT
CTTTCCAAC-3′
HGH17：5′-AATTGTTGGGAAAGATCCACCACCGCCGGATCCACCG
CCACCTAAGCC-3′
HGH18：5′-TTAGGCTTAGGCGGTGGTGGATCTGGTGGCGGCGGAT
CTGGTGGCGGTGGATCCTTCCCAAC-3′
HGH19：5′-AATTGTTGGGAAGGATCCACCGCCACCAGATCCGCC
GCCACCAGATCCACCACCGCCTAAGCC-3′
The annealing that HGH 16 and HGH are 17 causes n=2, and the annealing that HGH 18 and HGH are 19 then causes n=3.After the annealing, form pHGH56 (n=2 this moment) and pHGH57 (n=3 at this moment) among the pHGH10 that double chain oligonucleotide and the EcoRI-Bsu36I fragment cloning that comes from pHGH1 are cut to the Bsu36I enzyme (Figure 10).Correspondingly, the NotI expression cassette of these plasmids is cloned among the pSAC35 that the NotI enzyme cuts and forms pHGH58 and pHGH59 respectively.
As shown in Figure 11, introduced a BamHI site in the oligonucleotide of formation pHGH56 and pHGH57 clone's the joint sequence.Thereby can link on the BamHI-Hind III fragment that comes from pHGH57 (making n=1) by the Hind III-BamHI fragment that will come from pHGH56, the Hind III-BamHI fragment that maybe will come from pHGH57 is linked on the BamHI-HindIII fragment that comes from pHGH56 (making n=2) to make up joint sequence, n=1 and n=4 in this joint sequence.Be cloned into as described in these fragments such as the embodiment 2 in the Hind III site of pHA2, form pHGH60 (n=1) and pHGH61 (n=4) [seeing Figure 11] respectively.The NotI expression cassette that comes from pHGH60 and pHGH61 is cloned among the pSAC35 that the NotI enzyme cuts and is formed pHGH62 and pHGH63 respectively.
Use pHGH58, pHGH59, pHGH62 and pHGH63 transformed saccharomyces cerevisiae form the transformant that fusion polypeptide can be secreted in the supernatant liquor.
Utilize the existence of two component parts in the Western trace evidence fusion rotein that anti-HSA and anti-hGH antiserum(antisera) carry out.
Successively by cation-exchange chromatography, anion-exchange chromatography and gel permeation chromatography are with purified fusion protein for culture supernatant.This n-end of albumen of amino acid sequence analysis has proved the existence of the albumin sequence of expection.Analyze the albumen proof of this purifying compares with the HSA-hGH fusion rotein of description among the embodiment 3 by electrospray mass spectroscopy (electrospray mass spectrometry), this protein mass has increased 315D (n=1) as expecting, 630D (n=2), 945D (n=3) and 1260D (n=4).This purifying protein has activity in the experiment in vitro.
Reference Cunningham, B.C. etc. (1991) science (Science) 254,821-825.De Vos, A.M. etc. (1992) science (Science) 255,306-312.Ealey etc. (1995) growth regulating (Growth Regulation) 5,36-44.Gleeson etc. (1986) microbial genetics magazines (J.Gen.Microbiol) 132,3459-3465.Haffner, the clinical investigation magazines of D. etc. (1994) (J.Clin.Invest.93.1163-1171).Hiramitsu etc. (1990) applied environment thing biologies (App.Env.Microbiol.) 56,2125-2132.Hiramitsu etc. (1991) international bibliography: information and document (ibid) 57,2052-2056.Hoffman and Winston etc. (1990) heredity (Genetics) 124,807-816.Kearns, the clinical endocrine metabolism magazines of G.L. etc. (1991) (J.Clin.Endocrinol.Metab.) 72,1148-1156.Kunkel, T.A. etc. (1987) Enzymology method (Methods in Enzymol.) 154,367-382.Martial, J.A. etc. (1979) science (Science) 205,602-607.Maundrell (1990) journal of biological chemistry (J.Biol.Chem.) 265,10857-10864.Nomura, N. etc. (1995) bio-science, biotechnology, and biological chemistry (Biosci.Biotech.Biochem.) 59,532-534.Poznansky, the European biological chemistry of M.J. etc. (1988) association's federation's communication (FEBS lett.) 239,18-22.Saiki etc. (1988) science (Science) 239,487-491.Sambrook, (1989) molecular cloning (Molecular Cloning) such as J.: experiment guide (a LaboratoryManual), second edition (2nd edition), (the Cold Spring HarborLaboratory Press of press of cold spring harbor laboratory, Cold), the cold spring port (Spring Harbor, NY.).Sleep. wait (1990) biology and biotechnology (Bio/Technology) 8,42-46.Strobl, J.S. and Thomas, M.J. etc. (1994) pharmacology summary (Pharmacol.Rev.) 46,1-34.Tokunage, T. etc. (1985) genes (Gene) 39,117-120.Tsiomenko, A.B. etc. (1994) biological chemistries (Moscow) are (Moscow) 59 (Biochemistry), 1247-1256.Zeisel, H.J. etc. (1992) internal secretion summary (Horm.Res.) 37 supplementary issues 2 (Suppl.2), 5-13.
Claims (21)
1. polypeptide of forming by an amino acid successive zone that links together by peptide bond, comprise with an equal length district of tethelin (GH) at least tool 75% sequence homology at least ten amino acid whose first districts and have at least ten amino acid whose second districts of 75% sequence homology at least with serum albumin one equal length zone.
2. according to the polypeptide of claim 1, wherein each described first district and second district have 95% sequence homology respectively at least with the GH of described length and albumin.
3. according to a polypeptide of claim 2, each described first district and second district 50 amino acid longs of tool at least wherein.
4. according to the polypeptide of arbitrary aforementioned claim, wherein first district is by the uninterrupted amino acid of the C-end of GH, or its conservative property modified outcome forms, and this polypeptide in conjunction with and activated GH acceptor.
5. according to a polypeptide of claim 4, wherein first district is 191 amino acid longs.
6. according to a polypeptide of arbitrary aforementioned claim, albumin wherein and/or tethelin are the people sources.
7. according to a polypeptide of arbitrary aforementioned claim, wherein second district has at least one albuminous uninterrupted structural domain, or its conservative property modified outcome.
8. according to a polypeptide of claim 7, wherein the 1-105 of human albumin, 120-194,195-291,316-387 are contained in second district, 388-491,512-585,1-194,195-387,388-585,1-387, or the uninterrupted amino acid of 195-585, or its conservative property modified outcome.
9. according to a polypeptide of arbitrary aforementioned claim, wherein the N-end of this polypeptide contains said first district, and the C-end contains said second district.
10. the polypeptide arbitrary according to claim 1-8, wherein the N-end of this polypeptide contains said second district, and the C-end contains said first district.
11. a polypeptide according to arbitrary aforementioned claim contains said first district and second district, randomly the arbitrary end at this polypeptide adds amino acid or other compound again.
12. a microbiological culture media, it contains cell transformed and according to the polypeptide of arbitrary aforementioned claim.
13. a microbiological culture media has according to polypeptide arbitrary in the claim 1 to 11.
14. a coding is according to the polynucleotide of polypeptide arbitrary in the claim 1 to 11.
15. a microorganism host has the polynucleotide of being convenient to express among the host according to claim 14.
16. according to a host of claim 15, wherein polypeptide is secreted by the host.
17. be used for obtaining method, comprise and cultivate a kind of host and this polypeptide of purifying according to claim 15 or 16 according to the arbitrary polypeptide of claim 1 to 11.
18. a pharmaceutical preparation contains polypeptide arbitrary in the with good grounds claim 1 to 11 and medicine acceptable carrier.
19. a treatment suffers from patient's the method for the disease of available growth hormone therapy, this method comprises using to patient alleviates the said state of an illness, be not enough to the toxigenicity amount according to polypeptide arbitrary in the claim 1 to 11.
20. one kind is improved the method that the growth ratio of lean meat/fat meat in the animal body makes it to surpass normal value, comprise to animal use significant quantity according to polypeptide arbitrary in the claim 1 to 11.
21. method of producing tethelin, be included in a kind of microorganism host, preferably in yeast, expression is according to polynucleotide of claim 14, a this encoded polypeptides thereby site is arranged in described first district and second interval, this site can be cut by a kind of enzyme among the host, or by any other enzyme, or the chemical process cutting.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB9526733.2A GB9526733D0 (en) | 1995-12-30 | 1995-12-30 | Fusion proteins |
| GB9526733.2 | 1995-12-30 |
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| CN03110364A Division CN100590133C (en) | 1995-12-30 | 1996-12-19 | Growth hormone and serum albumni recombinant fusion protein |
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| CN03110364A Expired - Lifetime CN100590133C (en) | 1995-12-30 | 1996-12-19 | Growth hormone and serum albumni recombinant fusion protein |
| CN96199466A Expired - Lifetime CN1119421C (en) | 1995-12-30 | 1996-12-19 | Recombinant fusion proteins to growth hormone and serum albumin |
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| US (3) | US7045318B2 (en) |
| EP (2) | EP0870039B1 (en) |
| JP (1) | JP4063878B2 (en) |
| KR (1) | KR19990076789A (en) |
| CN (2) | CN100590133C (en) |
| AT (2) | ATE319838T1 (en) |
| AU (1) | AU715210B2 (en) |
| CA (1) | CA2240292C (en) |
| DE (2) | DE69637743D1 (en) |
| DK (2) | DK0870039T3 (en) |
| ES (2) | ES2318418T3 (en) |
| GB (1) | GB9526733D0 (en) |
| HK (1) | HK1066816A1 (en) |
| PT (2) | PT1681304E (en) |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101200503B (en) * | 2001-08-10 | 2010-07-07 | 中国人民解放军军事医学科学院生物工程研究所 | Fusion protein for seralbumin and interferon |
| CN1980687B (en) * | 2004-02-09 | 2015-05-13 | 人类基因科学公司 | Albumin fusion proteins |
| CN107949638A (en) * | 2015-09-08 | 2018-04-20 | Jcr制药股份有限公司 | New human serum albumins mutant |
| CN107949638B (en) * | 2015-09-08 | 2022-05-13 | Jcr制药股份有限公司 | Novel human serum albumin mutants |
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